The physiological basis of many seasonal allergies is the overproduction in the allergic patient of the allergic antibody, termed immunoglobulin E (IgE). This antibody is produced by specialized immune cells called B cells. IgE antibody can be over-produced in response to allergens (antigens) inhaled by allergic patients and this excess antibody combines with the allergen in the upper airways leading to the symptoms of allergy, such as itchy, watery eyes, runny nose, and even asthma. Understanding the basis for the overproduction of IgE in allergic patients is the goal of many studies by pharmaceutical companies. It is believed that by a deeper understanding of the basis of the allergic response, more effective therapies can be devised for prevention of the over-response. A central question in this area involves an understanding of the molecular mechanisms surrounding immunoglobulin (Ig) class switching in B cells (P. Takhar et al., Journal of Immunology, 2005, 174:5024-5032) from protective IgG antibodies to allergic IgE antibodies.
The peripheral cannabinoid receptor (CB2) was first identified in 1993 by Munro et al. (Nature, 365:61-65) via cloning of a novel G-protein coupled receptor expressed in the human cell line, HL60, which bound to cannabinoids with relatively high affinity. Since its discovery, the expression of CB2 is shown to be almost exclusively on cells of the immune system, with the ranking order of abundance being B cells>NK cells>macrophages>T cells (Galieque et al., Eur. J. Biochem., 1995, 232:54-61; Carayon et al., Blood, 1998, 92:3605-3615; Lee et al., Eur. J. Pharmacol., 2000, 423:235-241). Though B cells appear to express more CB2 receptor than other immune cell types, little is known about the role this receptor plays in B cell activation and overall biology. There is some evidence that CB2 signaling may be involved in B cell differentiation, migration, proliferation and antibody class switching, suggesting the receptor is a part of the B cell immune activation program (Carayon et al., Blood, 1998, 92:3605-3615; Jorda et al., Blood, 2002, 99:2786-2793; Rayman et al., J Immunol, 2004, 172:2111-2117; Massi et al., J Neuroimmunol, 1998, 92(1-2):60-6; Marchand et al., Cytometry, 1999, 35(3):227-34; Jorda et al., Ann N Y Acad. Sci., 2003, 996:10-6; Agudelo et al., Journal of NeuroImmune Pharmacology, 2008, 3:35-42). However, the mechanisms involved in CB2 receptor gene (CNR2) regulation and at what stage in B cell activation this process occurs have been unclear. Identifying the structure of the CNR2 gene promoter and the spectrum of transcription factors involved in gene regulation will help elucidate the role of CB2 in B cell biology. In this regard, several reports suggest that CD40 on B cells as well as stimulation by IL-4, and activation of STAT6, increases CB2 expression; whereas, LPS stimulation reportedly suppresses CB2 mRNA expression in B cells (Carayon et al., Blood, 1998, 92:3605-3615; Lee et al., Eur. J. Pharmacol., 2000, 423:235-241; Schroder et al., J Immunol, 2002, 168:996-1000; Agudelo et al., Journal of NeuroImmune Pharmacology, 2008, 3:35-42; Lee et al., Adv Exp Med Biol, 2001, 493:223-228). Though these studies provide some clues to regulation of CB2 expression in activated B cells, more studies are needed to define the factors that regulate CNR2 gene expression and CB2 protein in normal B cells.
The core promoter is the minimal region of DNA required for RNA polymerase II (Pol II) to assemble with the general transcription factors and form the pre-initiation complex for initiation of activator-independent (basal) transcription (Gross and Oelgeschlager, Biochem Soc Symp, 2006, 73:225-236). At the center of the core promoter is the initiator (INR) sequence that contains the transcription start site (TSS), which is defined as the most 5′ nucleotide of mRNA transcribed by Pol II (Gross and Oelgeschlager, Biochem Soc Symp, 2006, 73:225-236; Sandelin et al., Nat Rev Genet, 2007, 8:424-436). Correct identification of the TSS in primary resting B cells will lead to the location of the CNR2 core promoter, including core and cis-acting elements, and provide insights into the molecular mechanisms involved in CB2 expression.